CyHV-3感染镜鲤选育世代免疫基因表达及抗病能力比较
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摘要
对镜鲤抗锦鲤疱疹病毒(CyHV-3)新品种的选育工作,目前已经进行到F_3,F_2、F_3成活率相近且显著高于F_1。本研究利用实时荧光定量PCR技术比较选育世代间免疫基因IL-1β、TRAF6、My D88a、I-IFN和TLR7a的表达及Cy HV-3病毒载量情况,从而在免疫基因表达水平及病毒载量两个方面结合抗病成活率综合评估选育世代的抗病能力。结果显示:(1)Cy HV-3病毒载量在F_1、F_2、F_3中呈下降趋势,且F_3显著低于F_1、F_2(P<0.05);(2)夏花组未感染Cy HV-3,仅TRAF6基因在F_3的表达显著高于F_1、F_2(P<0.05);(3)感染组F_3脾中IL-1β、My D88a基因显著高于F_1、F_2(P<0.05)、TRAF6显著高于F_1(P<0.05),F_3肾中IL-1β、TRAF6基因显著高于F_1、F_2(P<0.05),F_1与F_2间均差异不显著(P>0.05)。以上结果说明在选育世代间除TRAF6基因表达较高外,其他基因未受选育影响,且F_3抗病能力显著高于F_1、F_2。基于F_2、F_3成活率相近且显著高于F_1,说明F_2抗病力不稳定,而F_3抗病力已趋于稳定,也说明从免疫基因表达或病毒载量方法进行抗病能力评估是可行的。
The mirror carp breeding of generations targeted at Koi herpes virus( Cy HV-3) has been carried out to F_3 generation. F_2 and F_3survival rates are similar and significantly higher than F_1. In this study,the Cy HV-3 viral loads and the gene expression of IL-1β,TRAF6,My D88 a,I-IFN and TLR7 a are compared by real-time fluorescence quantitative PCR technique. Immune gene expression levels and viral loads in combination with disease resistance survival rates are used to evaluate the disease resistance of different generations. The results showed that:( 1) Cy HV-3 virus load decreased sequentially in F_1,F_2 and F_3,and F_3 was significantly lower than F_1 and F_2( P < 0. 05);( 2) Summerlings group wasn't infected Cy HV-3,only the expression of TRAF6 gene in F_3 was significantly higher than that in F_1 and F_2( P < 0. 05).( 3) In the infected group,the levels of IL-1β and My D88 a in F_3 spleen were significantly higher than those in F_1 and F_2( P < 0. 05),and TRAF6 was significantly higher than that of F_1( P < 0. 05). The expression of IL-1β and TRAF6 in F_3 kidney was significantly higher than that in F_1 and F_2( P < 0. 05). There was no significant difference between F_1 and F_2( P > 0. 05). The above results show that F_3 disease resistance is significantly higher than F_1,F_2,only the TRAF6 gene was breeding. Based on the fact that F_2,F_3 survival rate was similar and significantly higher than F_1,it can be concluded that F_2 disease resistance is not stable and F_3 resistance has stabilized,and that using the immune gene expression or viral load method to evaluate the disease resistance is feasible.
The mirror carp breeding of generations targeted at Koi herpes virus( Cy HV-3) has been carried out to F_3 generation. F_2 and F_3survival rates are similar and significantly higher than F_1. In this study,the Cy HV-3 viral loads and the gene expression of IL-1β,TRAF6,My D88 a,I-IFN and TLR7 a are compared by real-time fluorescence quantitative PCR technique. Immune gene expression levels and viral loads in combination with disease resistance survival rates are used to evaluate the disease resistance of different generations. The results showed that:( 1) Cy HV-3 virus load decreased sequentially in F_1,F_2 and F_3,and F_3 was significantly lower than F_1 and F_2( P < 0. 05);( 2) Summerlings group wasn't infected Cy HV-3,only the expression of TRAF6 gene in F_3 was significantly higher than that in F_1 and F_2( P < 0. 05).( 3) In the infected group,the levels of IL-1β and My D88 a in F_3 spleen were significantly higher than those in F_1 and F_2( P < 0. 05),and TRAF6 was significantly higher than that of F_1( P < 0. 05). The expression of IL-1β and TRAF6 in F_3 kidney was significantly higher than that in F_1 and F_2( P < 0. 05). There was no significant difference between F_1 and F_2( P > 0. 05). The above results show that F_3 disease resistance is significantly higher than F_1,F_2,only the TRAF6 gene was breeding. Based on the fact that F_2,F_3 survival rate was similar and significantly higher than F_1,it can be concluded that F_2 disease resistance is not stable and F_3 resistance has stabilized,and that using the immune gene expression or viral load method to evaluate the disease resistance is feasible.
引文
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[15]包卫空,沈锦玉.锦鲤疱疹病毒的鉴定及预防[J].水产养殖,2015,36(4):45-48.BAO W K,SHEN J Y.Identification and prevention of Koi herpes virus disease[J].Journal of Aquaculture,2015,36(4):45-48.
[16]φDEGARD J,OLESEN I,DIXON P,et al.Genetic analysis of common carp(Cyprinus carpio)strains.II:Resistance to koi herpesvirus and Aeromonas hydrophila and their relationship with pond survival[J].Aquaculture,2010,304(1/4):7-13.
[17]张圆圆,王先科,贾滔.锦鲤疱疹病毒(KHV)的检测技术概述[J].河南水产,2016(4):1-2,6.ZHANG Y Y,WANG X K,JIA T.Overview of detection methods of koi herpesvirus(KHV)[J].Henan Shuichan,2016(4):1-2,6.
[18]ADAMEK M,SYAKURI H,HARRIS S,et al.Cyprinid herpesvirus 3 infection disrupts the skin barrier of common carp(Cyprinus carpio L.)[J].Veterinary Microbiology,2013,162(2/4):456-470.
[19]张艳,孟庆峰,钱爱东,等.锦鲤疱疹病毒荧光定量PCR检测方法的建立[J].吉林畜牧兽医,2010,31(4):11-14.ZHANG Y,MENG Q F,QIAN A D,et al.Establishment of a flurogenic quantitative polymerase chain reaction assay for detection of Koi hepesvirus[J].Jilin Animal Husbandry and Veterinary Medicine,2010,31(4):11-14.
[20]葛会争.德国镜鲤与黑龙江野鲤杂交组合的亲子鉴定及鲤鱼免疫功能基因SNP位点研究[D].上海:上海海洋大学,2012.GE H Z.Parentage identification of Heilongjiang wild carp and the German mirror carp cross combinations on microsatellite DNA markers and carp immune function gene SNP research[D].Shanghai:Shanghai Ocean University,2012.
[21]LIVAL K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and themethod[J].Methods,2001,25(4):402-408.
[22]XU Q Q,XU P,ZHOU J W,et al.Cloning and expression analysis of two pro-inflammatory cytokines,IL-1βand its receptor,IL-1R2,in the Asian swamp eel Monopterus albus[J].Molecular Biology,2016,50(5):671-683.
[23]刘佳佳,吴志新,石焱,等.甘露寡糖对草鱼感染嗜水气单胞菌后细胞因子表达的调节[J].淡水渔业,2013,43(5):31-36.LIU J J,WU Z X,SHI Y,et al.Regulation of MOS on cytokines gene expression in grass carp infected with Aeromonas hydrophila[J].Freshwater Fisheries,2013,43(5):31-36.
[24]VEKSLER-LUBLINSKY I,SHEMER-AVNI Y,MEIRI E,et al.Finding quasi-modules of human and viral miRNAs:a case study of human cytomegalovirus(HCMV)[J].BMC Bioinformatics,2012,13:322.
[25]JENKINS K A,MANSELL A.TIR-containing adaptors in Toll-like receptor signalling[J].Cytokine,2010,49(3):237-244.
[2]ADANEK M,STEINHAGEN D,IRNAZAROW I,et al.Biology and host response to Cyprinid herpesvirus 3 infection in common carp[J].Developmental&Comparative Immunology,2014,43(2):151-159.
[3]RAKUS K L,IRNAZAROW I,ADAMEK M,et al.Gene expression analysis of common carp(Cyprinus carpio L.)lines during Cyprinid herpesvirus 3 infection yields insights into differential immune responses[J].Developmental&Comparative Immunology,2012,37(1):65-76.
[4]KONGCHUM P,PALTI Y,HALLERMAN E M,et al.SNP discovery and development of genetic markers for mapping innate immune response genes in common carp(Cyprinus carpio)[J].Fish&Shellfish Immunology,2010,29(2):356-361.
[5]KONGCHUM P,HALLERMAN E M,HULATA G,et al.Molecular cloning,characterization and expression analysis of TLR9,My D88 and TRAF6 genes in common carp(Cyprinus carpio)[J].Fish&Shellfish Immunology,2011,30(1):361-371.
[6]杨华,杨永林.基于主成分分析法建立绵羊一般抗病力的评估模型[J].中国草食动物科学,2015,35(3):1-5.YANG H,YANG Y L.Assessment model of general disease resistance with principal component analysis in sheeps[J].China Herbivore Science,2015,35(3):1-5.
[7]梁忠秀,李健,谭志军,等.塔玛亚历山大藻对中国明对虾肝胰腺和鳃SOD、GST和MDA的影响[J].水产学报,2013,37(8):1192-1197.LIANG Z X,LI J,TAN Z J,et al.Effects of the toxic dinoflagellate Alexandrium tamarense on MDA,SOD and GST in hepatopancreas and gill of Fenneropenaeus chinensis[J].Journal of Fisheries of China,2013,37(8):1192-1197.
[8]SHIN G W,WHITE S L,DAHMS H U,et al.Disease resistance and immune-relevant gene expression in golden mandarin fish,Siniperca scherzeri Steindachner,infected with infectious spleen and kidney necrosis virus-like agent[J].Journal of Fish Diseases,2014,37(12):1041-1054.
[9]AHMADI P Y,FARAHMAND H,MIANDARE H K,et al.The effects of dietary Immunogenon innate immune response,immune related genes expression and disease resistance of rainbow trout(Oncorhynchus mykiss)[J].Fish&Shellfish Immunology,2014,37(2):209-214.
[10]李俊超,马杰,曾令兵,等.抗锦鲤疱疹病毒的植物药物筛选及其效果比较[J].淡水渔业,2014,44(3):62-67.LI J C,MA J,ZENG L B,et al.Studies on the screening and efficacy evaluation of herbal drugs in inhibiting Koi herpesvirus replication in vitro[J].Freshwater Fisheries,2014,44(3):62-67.
[11]袁海延,于慧,王好,等.锦鲤疱疹病毒病的研究进展[J].中国兽药杂志,2015,49(5):62-65.YUAN H Y,YU H,WANG H,et al.Research progress of koi herpesvirus disease[J].Chinese Journal of Veterinary Drug,2015,49(5):62-65.
[12]贾智英,池喜峰,李池陶,等.德国镜鲤养殖群体中抗病与死亡个体遗传结构比较研究[J].上海海洋大学学报,2010,19(1):7-11.JIA Z Y,CHI X F,LI C T,et al.Analysis of genetic structure between the death and survival individuals of German mirror carp in cultivated stocks[J].Journal of Shanghai Ocean University,2010,19(1):7-11.
[13]朱霞,李新伟,王好,等.一株锦鲤疱疹病毒的分离与鉴定[J].中国预防兽医学报,2011,33(5):340-343.ZHU X,LI X W,WANG H,et al.Isolation and identification of Koi herpesvirus[J].Chinese Journal of Preventive Veterinary Medicine,2011,33(5):340-343.
[14]周永灿,袁军法,任武泽,等.锦鲤疱疹病毒的检测与人工感染试验[J].武汉大学学报(理学版),2005,51(s2):249-252.ZHOU Y C,YUAN J F,REN W Z,et al.Detection and experimental infection of Koi herpesvirus[J].Journal of Wuhan University(Natural Science Edition),2005,51(s2):249-252.
[15]包卫空,沈锦玉.锦鲤疱疹病毒的鉴定及预防[J].水产养殖,2015,36(4):45-48.BAO W K,SHEN J Y.Identification and prevention of Koi herpes virus disease[J].Journal of Aquaculture,2015,36(4):45-48.
[16]φDEGARD J,OLESEN I,DIXON P,et al.Genetic analysis of common carp(Cyprinus carpio)strains.II:Resistance to koi herpesvirus and Aeromonas hydrophila and their relationship with pond survival[J].Aquaculture,2010,304(1/4):7-13.
[17]张圆圆,王先科,贾滔.锦鲤疱疹病毒(KHV)的检测技术概述[J].河南水产,2016(4):1-2,6.ZHANG Y Y,WANG X K,JIA T.Overview of detection methods of koi herpesvirus(KHV)[J].Henan Shuichan,2016(4):1-2,6.
[18]ADAMEK M,SYAKURI H,HARRIS S,et al.Cyprinid herpesvirus 3 infection disrupts the skin barrier of common carp(Cyprinus carpio L.)[J].Veterinary Microbiology,2013,162(2/4):456-470.
[19]张艳,孟庆峰,钱爱东,等.锦鲤疱疹病毒荧光定量PCR检测方法的建立[J].吉林畜牧兽医,2010,31(4):11-14.ZHANG Y,MENG Q F,QIAN A D,et al.Establishment of a flurogenic quantitative polymerase chain reaction assay for detection of Koi hepesvirus[J].Jilin Animal Husbandry and Veterinary Medicine,2010,31(4):11-14.
[20]葛会争.德国镜鲤与黑龙江野鲤杂交组合的亲子鉴定及鲤鱼免疫功能基因SNP位点研究[D].上海:上海海洋大学,2012.GE H Z.Parentage identification of Heilongjiang wild carp and the German mirror carp cross combinations on microsatellite DNA markers and carp immune function gene SNP research[D].Shanghai:Shanghai Ocean University,2012.
[21]LIVAL K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and themethod[J].Methods,2001,25(4):402-408.
[22]XU Q Q,XU P,ZHOU J W,et al.Cloning and expression analysis of two pro-inflammatory cytokines,IL-1βand its receptor,IL-1R2,in the Asian swamp eel Monopterus albus[J].Molecular Biology,2016,50(5):671-683.
[23]刘佳佳,吴志新,石焱,等.甘露寡糖对草鱼感染嗜水气单胞菌后细胞因子表达的调节[J].淡水渔业,2013,43(5):31-36.LIU J J,WU Z X,SHI Y,et al.Regulation of MOS on cytokines gene expression in grass carp infected with Aeromonas hydrophila[J].Freshwater Fisheries,2013,43(5):31-36.
[24]VEKSLER-LUBLINSKY I,SHEMER-AVNI Y,MEIRI E,et al.Finding quasi-modules of human and viral miRNAs:a case study of human cytomegalovirus(HCMV)[J].BMC Bioinformatics,2012,13:322.
[25]JENKINS K A,MANSELL A.TIR-containing adaptors in Toll-like receptor signalling[J].Cytokine,2010,49(3):237-244.